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Activation of the epidermal growth factor signalling pathway by tissue plasminogen activator in pancreas cancer cells
  1. Mariano Hurtado1,
  2. Juan José Lozano2,*,
  3. Elisabeth Castellanos1,
  4. Luis A López-Fernández3,
  5. Keith Harshman3,
  6. Carlos Martínez-A3,
  7. Angel R Ortiz2,
  8. Timothy M Thomson4,
  9. Rosanna Paciucci1
  1. 1Unitat de Recerca Biomedica, Institut de Recerca, Hospital Vall d’Hebrón, Barcelona, Spain
  2. 2Unidad de Bioinformática, Centro de Biología Molecular “Severo, Ochoa”, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain
  3. 3Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain
  4. 4Departament de Biología Molecular i Celular, Institut de Biología Molecular-Consejo Superior de Investigaciones Científicas, Barcelona, Spain
  1. Correspondence to:
    Rosanna Paciucci
    Unitat de Recerca Biomédica, Institut de, Recerca Hospital Vall d’Hebrón, Pg Vall d’Hebrón 119-129, Barcelona 08035, Spain; rpaciucci{at}ir.vhebron.net

Abstract

Background: Tissue plasminogen activator (tPA) is the major activator of plasminogen in plasma. This serine protease is overexpressed by exocrine pancreas tumour cells, where it promotes tumour cell proliferation, growth, and invasion. Here we have explored the signalling pathways used by tPA to activate the proliferation of pancreatic cancer cells.

Methods: Transcriptional profiling on cDNA micro arrays was used to analyse the pattern of gene expression in response to tPA compared to the response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Results were confirmed using different biochemical assays in which specific kinase inhibitors or RNA interference were used.

Results: Transcriptional profiling showed that tPA modulates the expression of a set of genes commonly regulated by EGF, but distinct from the major set of genes modulated by PDGF. This suggested that tPA and EGF share common signalling pathways, a conclusion supported by further experimental evidence. Firstly, we found that tPA induced a rapid and transient phosphorylation of the EGFR. Secondly, specific EGFR kinase inhibitors, but not PDGFR kinase inhibitors, abolished the tPA induced phosphorylation of the ERK1/2 kinases and cell proliferation. The mitogenic activity of tPA was also inhibited by siRNA depletion of EGFR, thus confirming the involvement of this receptor in tPA triggered signalling. Thirdly, we show that the signalling and mitogenic effects of tPA require its proteolytic activity, the activity of the metalloprotease-9 and active hb-EGF.

Conclusion: Our results suggest that tPA induces proliferation by triggering a proteolytic cascade that sequentially activates plasmin, metalloprotease-9 (MMP-9) and hb-EGF. These events are required to activate the EGFR signalling pathway and cell proliferation.

  • DFP, diisopropyl fluorophosphate
  • DMEM, Dulbecco’s Modified Eagle’s Medium
  • EGFR, epidermal growth factor receptor
  • ERK 1/2, extracellular activated kinases 1 and 2
  • FA, factor analysis
  • FBS, fetal bovine serum
  • hb-EGF, heparin binding-epidermal growth factor
  • MMP-9, metalloprotease-9
  • PAs, plasminogen activators
  • PCR, polymerase chain reaction
  • PDGF, platelet derived growth factor
  • rtPA, recombinant tPA
  • siRNA, small interfering RNA duplexes
  • TGFα, transforming growth factor α
  • tPA, tissue type plasminogen activator
  • uPA, urokinase type plasminogen activator
  • tissue plasminogen activator
  • pancreas cancer
  • EGF receptor
  • proliferation
  • Microarrays

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Footnotes

  • * Present address: Center for Genome Regulation, Barcelona, Spain

  • Published Online First 23 April 2007

  • Funding: This work was supported by grants from former Ministerio de Educación (SAF2000-0195), Fondos de Investigación Sanitaria (PI020764), Fundació Marató-TV3 and Hospital Vall d’Hebrón (to RP), grant GEN2001-4856-C13-07 and GEN2001-4856-C13-10 from the former Ministerio de Ciencias y Tecnología (to TMT and ARO, respectively). MH was a recipient of a fellowship from the Institut de Recerca Hospital Vall d’Hebrón.

  • Competing interests: none.

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